Systemic autoimmunity in BAFF-R-mutant A/WySnJ strain mice

Abstract

Systemic lupus erythematosis is an autoimmune disease of unknown etiology. Lupus pathology is thought to reflect autoantibody-mediated damage due to a failure of B lymphocyte tolerance. Since excessive B cell-activating factor belonging to the TNF family (BAFF) expression correlates with human and murine lupus, and BAFF signals B cell survival through BAFF-R, it is believed that excessive BAFF-R signaling can subvert B cell tolerance and facilitate lupus development. Here we report the unexpected finding that BAFF-R-mutant A/WySnJ mice develop a lupus-like syndrome. These mice carry the B cell maturation defect-1 (Bcmd-1) mutant allele of the Baffr gene. Bcmd-1 causes premature B cell death and profound B cell deficiency. Despite having 90% fewer splenic B cells than normal mice, A/WySnJ mice had an 18-fold increased frequency of splenocytes secreting IgM antibodies to dsDNA, and increased amounts of circulating IgM and IgG to dsDNA by 9 months of age. By age 11 months, most A/WySnJ mice displayed renal pathology characteristic of lupus, including proteinuria as well as periodic acid-Schiff-positive deposits and glomerular capillary bed destruction. Importantly, we genetically linked this autoimmunity to Bcmd-1, since congenic AW.Baffr(+/+) mice carrying a wild-type allele developed none of these phenotypes. Our data provide the first evidence linking altered BAFF-R signaling to the development of B cell-mediated autoimmunity.

Figure 1

A/WySnJ mice produce autoantibodies to dsDNA, whereas A/J and AW.Baffr^+/+ mice do not. (A) IgM autoantibodies to dsDNA. (B) IgG autoantibodies to dsDNA. (C) IgM AFC specific for dsDNA. Serum autoantibodies to dsDNA were quantified by ELISA. AFC were quantified by ELISPOT assay. The ELISA data shown are the mean absorbance (± SD) for six to nine male and female mice at 7–9 months of age. The ELISPOT data shown are the mean (± SD) for six to 12 mice ≥9 months of age. The Mann–Whitney test and two-tailed Student’s t-test gave p<0.01 (*) for comparisons between the A/WySnJ and the A/J or AW.Baffr^+/+ mice.

Figure 2

A/WySnJ mice are B lymphopenic, whereas A/J and congenic AW.Baffr^+/+ mice are not. (A) Schematic representation of Baffr cDNA. Gray shading represents the retrotransposon insertion. (B) AW.Baffr^+/+ chromosome 15. Gray shading shows the congenic interval derived from the CAST/Ei strain. (C) Splenocytes were stained with fluorescent antibodies to IgM and IgD, and analyzed for the T1 (IgM^highIgD^low), MZ/T1 (IgM^highIgD^int), T2 (IgM^highIgD^high), and mature B cell subsets (IgM^lowIgD^high). (D) Splenocytes were stained with fluorescent antibodies to IgM, C1qRp, and CD23. The IgM-gated cells were analyzed for the mature B cell subset (CD23^highC1qRp⁻). The C1qRp-gated cells were analyzed for T1 (IgM^highCD23⁻), T2 (IgM^highCD23⁺), and T3 (IgM^lowCD23⁺) B cell subsets. The data are representative of six mice per strain.

Figure 3

Retroviral-mediated gene transfer of the wild-type Baffr gene into A/WySnJ HSC-restored B cell development. Flow cytometry was performed on individual splenocyte samples gated according to their GFP expression. (A) The cells were stained with antibodies to IgM, CD21 and CD23, and gated for NF, MZ and FO B cell subsets. (B) The cells were stained with antibodies to IgM and to IgD; the IgM⁺IgD⁺ cells were gated as shown to evaluate B cell subsets. (C) The cells were stained with antibodies to IgM, C1qRp, and CD23; the IgM⁺ cells were gated for the C1qRp⁻CD23⁺ mature B cells [47]. (D) The cells were stained with antibodies to IgM, C1qRp, and CD23; the C1qRp⁺ cells were subdivided into T1, T2 and T3 B cells. The data shown are representative of five mice per strain.

Figure 4

A/WySnJ mice develop proteinuria, whereas A/J and AW.Baffr^+/+ mice do not. (A) Mean proteinuria score. (B) Incidence of severe proteinuria (≥300 mg/dL urinary protein). Scores were assigned as follows: score 0 = 0-trace, score 30 = 30–99 mg/dL, score 100 = 100–299 mg/dL, score 300 = ≥300 mg/dL. The data shown are from 13–22 female and male mice per age group. A two-tailed Student’s t-test for proteinuria scores and χ² test of incidence gave p<0.05 (*) for comparisons between A/WySnJ and A/J or AW.Baffr^+/+ mice.

Figure 5

A/WySnJ mice develop renal pathology and accumulate IgG deposits in the glomeruli, whereas A/J and AW.Baffr^+/+ mice do not. (A) Kidney sections stained with PAS reagent. (B) Kidney sections within the white boxes in panel (A) were enlarged to show renal pathology. (C) Kidney sections stained with FITC-coupled antibodies to mouse IgG. Histopathological and immunohistochemical analyses were performed on kidneys from mice aged 11–12 months.